Various labeling agents have hitherto been developed for a visualization or quantification of trace amounts of compounds. Radioisotopes such as tritium, radioiodine, and others have been used as typical labeling agents, particularly in fields that demand a high-sensitivity, and quantified, for example, by exposure to photographic film or a measurement of radioactivity in a scintillation counter.
More recently, enzyme immunoassays have been developed as a method not influenced by a restriction imposed by handling radioactive materials. Development of the methods for labeling antigens or antibodies such as persoxidase, alkaline phosphatase, glucose oxidase, β-D-galactosidase, or the like established a quantification method suitable for enzyme immunoassay or observation of sections of tissues by staining.
On the other hand, as a method for observing sections of tissues, the method is known of labeling antibodies with a fluorescent substance (for example, fluorescein, rhodamine, or the like), and observing under a fluorescence microscope after the reaction. This method has many advantages in that the use thereof is not restricted, as it does not use radioactive substances, and that there is no need for an additional step of adding a substrate as in the enzyme-based assays, but also has a drawback in that the absolutely sensitivity thereof is not high enough for use as a labeling assay.
One of the most advanced forms of the fluorescence labeling method is the time-resolved fluorescence measurement. This is a method in which the accuracy and sensitivity of measurement is raised by irradiating pulsed excitation light to fluorescent substances that have a long fluorescence quenching time, such as an europium chelate; measuring the fluorescence after a certain period of time when the direct excitation light and short-lived fluorescence derived from the surrounding substances have been quenched; and then measuring the fluorescence specific to europium.
An attempt to raise the sensitivity further by including this europium chelate in a polystyrene particle and thus increasing the amount of fluorescent substance per particle has been made. Although this method can raise the amount of fluorescence per particle, it also has a drawback in that the resulting particle has a hydrophobic surface, as polystyrene per se is hydrophobic, and thus, when used in biological reactions, it absorbs a large number of clingy hydrophobic substances present in the environment, and thus the method should be used while considering this disadvantage.
Alternatively for the purpose of pursuing convenience rather than raising sensitivity, an attempt was also made to visually detect polystyrene particles immobilized by antigen-antibody reaction, by preparing a reagent by including a dye in a polystyrene particle and coating an antigen or antibody on the surface of the polystyrene particle.
Even though the methods known in the art of making such labeling substances by including dye or fluorescent substances in a polystyrene particle are advantageous in that they are easy to operate or provide a significantly high sensitivity, they are still accompanied by occasional errors in determination due to nonspecific reactions, even after an attempt to overcome the disadvantages of using the polystyrene particles having a hydrophobic surface, for example, by: (1) after binding a desired functional molecule such as antigen, antibody, or the like, covering the hydrophobic surface of the polystyrene with a substance such as a protein or a biological substance-resembling substance by coating it on the non-bonded surface thereof; or (2) adding a surfactant to the reaction liquid and preventing a mutual interaction between polystyrene particles.
Accordingly, the methods of using a polystyrene particle as a carrier could not overcome the disadvantage in sensitivity and specificity, even when the particle was stabilized with a blocking agent or an additive was introduced into the buffer solution during reaction, to suppress non-specific reactions.